Activation of Adhesion G Protein-coupled Receptors by a Tethered Agonist: Mechanism of Action and Pharmacological Modulation

Thesis (Ph.D.)--University of Rochester. School of Medicine & Dentistry. Dept. of Pharmacology and Physiology, 2016.The recently defined adhesion G protein-coupled receptors (adhesion GPCRs) are a poorly characterized GPCR family with seven transmembrane topology and very large extracellular domains harboring numerous adhesion modules. These orphan receptors have profound therapeutic potential, with established roles in oncogenesis and metastasis. However, mechanistic understanding of adhesion GPCR activation and intracellular signaling is limited. Hence, efforts to discover pharmacological modulators for these proteins have been stunted. We have uncovered a common mechanism of activation that may apply to all 33 receptors. aGPCRs have an evolutionarily conserved GPCR Autoproteolysis Inducing (GAIN) domain that divides the receptor into two discrete portions: the extracellular domain (ECD) and the 7 transmembrane domain (7TM). The ECD and 7TM are noncovalently bound at the plasma membrane, positioned for activation. It is believed that extracellular matrix proteins bind the ECDs of these proteins, eliciting a removal/rearrangement. We discovered that for G protein receptors 56 and 110, the ECD sequesters an ~20 amino acid tethered agonist covalently bound to the 7TM and that upon ECD removal, the 7TM directly activates heterotrimeric G proteins. Using our established [35S]-GTPγS activation assay, we directly measured receptormediated G protein activation, proving that these receptors are bona fide GPCRs and defining the subtypes of G proteins to which GPRs 56 and 110 will couple. Having established the adhesion GPCR mechanism of activation, we performed luciferase gene reporter assays adapted for high throughput screening to identify inhibitors of the active (tethered agonist bound) GPR56 7TM receptor and activators of a low-activity (truncated tethered agonist) GPR56 7TM receptor. The isoflavonoid, dihydromunduletone, was the most efficacious GPR56 inhibitor while the natural product isolated from the Indian Neem tree, 3-alphaacetoxydihydrodeoxygedunin, was an efficacious GPR56 activator. Both compounds were able to respectively inhibit and activate other adhesion GPCRs including the related GPR114 but could not inhibit or activate other non-adhesion GPCRs, including the β2 adrenergic receptor and the M3 muscarinic receptor. Collectively, we have uncovered the adhesion GPCR activation mechanism and used it to identify an aGPCR inhibitor and an aGPCR activator that may be used for lead optimization and eventual therapeutic use

Structural Basis for Regulation of GPR56/ADGRG1 by Its Alternatively Spliced Extracellular Domains

Adhesion G-protein-coupled receptors (aGPCRs) play critical roles in diverse neurobiological processes including brain development, synaptogenesis, and myelination. aGPCRs have large alternatively spliced extracellular regions (ECRs) that likely mediate intercellular signaling; however, the precise roles of ECRs remain unclear. The aGPCR GPR56/ADGRG1 regulates both oligodendrocyte and cortical development. Accordingly, human GPR56 mutations cause myelination defects and brain malformations. Here, we determined the crystal structure of the GPR56 ECR, the first structure of any complete aGPCR ECR, in complex with an inverse-agonist monobody, revealing a GPCR-Autoproteolysis-Inducing domain and a previously unidentified domain that we term Pentraxin/Laminin/neurexin/sex-hormone-binding-globulin-Like (PLL). Strikingly, PLL domain deletion caused increased signaling and characterizes a GPR56 splice variant. Finally, we show that an evolutionarily conserved residue in the PLL domain is critical for oligodendrocyte development in vivo. Thus, our results suggest that the GPR56 ECR has unique and multifaceted regulatory functions, providing novel insights into aGPCR roles in neurobiology